Dysregulation of the WNK4-SPAK/OSR1 pathway has a minor effect on baseline NKCC2 phosphorylation

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na⁺-K⁺-2Cl^- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4^-/-, Osr1^-/-, Spak^-/-, and Osr1^-/-/Spak^-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3^+/-/Δ9, Klhl3^-/-, and Klhl3^R528H/R528H). All mice were on the C57BL/6 background. In Wnk4^-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak^-/- and Osr1^-/-/Spak^-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1^-/- and Osr1^-/-/Spak^-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3^-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na⁺ excretion following thiazide treatment was similar between Klhl3^-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt.NEW & NOTEWORTHY The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.